The present invention relates to a screening method for identifying analgesic compositions and to a novel class of analgesic agents.
Akopian, A. N., et al., Nature 379:257-262 (1996).
Arbuckle, J. B. and R. J. Docherty, Neurosci. Lett. 185:70-73 (1995).
Del-Bianco, E., et al., Neurosci. Lett. 170:163-166 (1994).
Elliott, A. A. and J. R. Elliott, J. Physiol. 463:39-56 (1993).
Fernando, C. R. et al., J. Med. Chem. 23:1153-1158 (1980).
Glushkov, R. G. et al., U.S. Pat. No. 1,557,964 (7/92); Derwent Abstract No. 93-224958.
Gold, M. S., et al., Proc. Natl. Acad. Sci. (USA) 93:1108-1112 (1996).
Hamill O. P., et al., Pflugers Arch. 391:85-100 (1981).
Jeftinija, S., Brain Research 639:125-134 (1994).
Keenaghan, J. B. et al., J. Pharmacol. Exp. Ther. 180:454 (1972).
Lawson, S. N., et al., Brain Res. Bull. 30:239-243 (1993).
Marsh, S. J., et al., Neuroscience 23:275-289 (1987).
Martin, H. A., et al., Neuroscience 22:651-659 (1987).
Nelson, S. D. et al., J. Pharrm. Sci. 66:1180-1190 (1977).
Oh, U., et al., J. Neurosci. 16:1659-1667 (1996).
Roy, M. L. and T. Narahashi, J. Neurosci. 12:2104-2111 (1992).
Treede, R. D., et al., Prog. Neurobiol. 38:397-421 (1992).
Treede, R. D., Annals of Medicine 27:213-216 (1995).
Chronic or intractable pain, such as may occur in conditions such as bone degenerative diseases and cancer, is a debilitating condition which is treated with a variety of analgesic agents, and often opioid compounds, such as morphine.
While brain pathways governing the perception of pain are still incompletely understood, sensory afferent synaptic connections to the spinal cord, termed xe2x80x9cnociceptive pathwaysxe2x80x9d have been documented in some detail. In the first leg of such pathways, C- and A-fibers are primary afferents which project from peripheral sites to the spinal cord, carrying nociceptive signals. Polysynaptic junctions in the dorsal horn of the spinal cord are involved in the relay and modulation of sensations of pain to various regions of the brain, including the periaqueductal grey region. Analgesia, or the reduction of pain perception, can be effected directly by decreasing transmission along such nociceptive pathways. Analgesic opiates are thought to act by mimicking the effects of endorphin or enkephalin peptide-containing neurons, which synapse presynaptically at the C- or A-fiber terminals and which, when they fire, inhibit release of neurotransmitters, including substance P. Descending pathways from the brain are also inhibitory on C- and A-fiber firing.
Mammalian dorsal root ganglia (DRG) contain several subtypes of primary afferent neurons. Afferent neurons in dorsal root ganglia receive input from axons that originate in the periphery and terminate in the dorsal horn of the spinal cord. These signals are carried by action potentials and are coded by the rate that action potentials invade synaptic termini in the dorsal horn. DRG neurons transmit information concerning muscle length and tension, touch, joint movement, temperature, as well as, fast and slow pain. DRG neurons that subserve the perception of pain are termed nociceptors or primary afferent nociceptors (PANs; Treede, 1995). PANs are distinguishable from other DRG neuron-subtypes by their small diameter axons (both myelinated and unmyelinated), a small diameter cell body (Lawson, et al., 1993) and by the presence of capsaicin-sensitive receptors that are coupled to non-selective cation channels (Marsh, et al., 1987; Martin, et al., 1987; Oh, et al., 1996). PANs become sensitized by sustained noxious stimuli and inflammatory mediators. Sensitization is manifested by a reduction in threshold and by a response increase (i.e. the firing rate of action potentials; Treede, 1992). The sensitization of PANs is thought to underlie hyperalgesia and may also contribute to some neuropathic pain states (Treede, 1995).
Blocking the signals carried by PANs leads to analgesia. Analgesia may be achieved by preventing the initiation of the signal, by blocking signal transmission along the PANs between the sensory ending and pre-synaptic terminal, or by blocking synaptic signal transmission in the dorsal horn of the spinal cord. For example, the local anesthetics are believed to block pain signals by blocking voltage-gated sodium (Na) channels in PANs and thus preventing the signal from reaching the presynaptic terminal.
DRG neurons that carry different types of sensory information transmit their signals using a number of voltage- and ligand-dependent ion channels. Of particular interest in the context of analgesia are the Na channel subtypes expressed in DRG and other neuron types. Na channels in PANs are dominated by a subtype that is insensitive to the neurotoxin tetrodotoxin (tetrodotoxin-insensitive (TTXi) Na channel; Akopian, et al., 1996; Arbuckle, et al., 1995; Gold, et al., 1996; Jeftinija, 1994). However, most neurons, such as motor neurons, also express significant levels of TTX-sensitive Na channels (TTXs Na channel; Elliot; Roy).
It would be desirable to selectively block Na channels enriched in PANs while avoiding the inhibition of Na channels expressed in other neuron subtypes, to reduce adverse side effects, while still effecting analgesia. For example, while lidocaine and other local anesthetics can act as potent analgesics, because they block TTXs Na channels, they can produce serious side effects, including spinal block, respiratory blockade and cardiac arrest. These compounds must be administered with great caution.
It is the discovery of the present invention that agents that preferentially block TTXi Na channels over TTXs Na channels are particularly useful as analgesic agents and represent a significant improvement over existing local anesthetics. We describe herein, a new class of agents, exemplified by SNX-483 (3-hydroxy monoethylglycinexylidide, 3-OH MEG-X), that selectively block TTXi Na channels in mammalian PANs, as well as a screening assay for identifying these and similar compounds.
In one aspect, the invention includes a method of selecting compounds for use in producing analgesia. The general method includes measuring the ability of test compounds to block tetrodotoxin-insensitive (TTXi) sodium channels. A compound is selected as an analgesic agent if it is at least{fraction (1/10)} as potent as 3-hydroxy monoethylglycinexylidide (3-OH-MEGX) in locking such channels. In another embodiment, the compound will be equipotent to 3-OH-MEGX in this locking activity.
The method includes further measuring the ability of the test compound to block tetrodotoxin-sensitive (TTXs) sodium channels. Desirable compounds selected by this embodiment of the method will be selected if they exhibit potency ratios of at least 2 in blocking TTXI sodium channels as compared to blocking TTXs sodium channels. In a preferred embodiment, the potency ratio for comparing blockade of TTXi and TTXs is calculated according to the formula:
Potency ratio=1(IC50, TTXi/IC50, TTXs),
where IC50, TTXi is the concentration of compound effective to inhibit current through the TTXi sodium channel by 50%, and IC50, TTXs is the concentration of compound effective to inhibit current through the TTXs sodium channel by 50%.
In another embodiment, the compound selection includes monitoring the compound for its ability to reversibly block said TTXi sodium channel in a cell bathing solution in an electrophysiological assay of TTXI sodium channels. A compound is selected if not more than 5-15% of a sodium current that passes through the TTXi sodium channel recovers after said compound is removed from the cell bathing solution. Such recovery is preferably measured over a time frame of 5-30 minutes of continuous perfusion of the cell preparation following removal of compound from the cell bathing solution.
In alternate embodiments, blockade of TTX-insensitive channels can be measured either by electrophysiological means, such as in the electrophysiological assays described herein, or by ligand binding displacement assays, also described herein. In such a ligand displacement, a preferred ligand is 3-hydroxy monoethylglycinexylidide (3-OH-MEGX).
In a related aspect, the invention includes a composition comprising a 3-hydroxy monoethylglycinexylidide analog that is effective to block TTXi sodium channels with a potency that is at least about one-tenth as great as the potency exhibited by 3-hydroxy monoethylglycinexylidide in blocking such TTXi sodium channels. More preferably, the composition will exhibit a potency that is at least as great as the potency exhibited by 3-hydroxy monoethylglycinexylidide in blocking TTX-insensitive channels. The compound is also characterized by a potency ratio in blocking TTXi sodium channels, as compared to blocking TTXs sodium channels, of at least 2, as described above.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.